The principal investigator has identified several bands in human chromosomes that appear to be among the earliest regions to be replicated in human cells. He has some evidence that he has cloned one of these early replicating regions. The goal of this work is to identify cosmid clones containing six of the regions that seem to have early replicating DNA (aim 1), show that these clones contain consensus sequences that are generally thought to be associated with the initiation of DNA replication (aim #2), and establish that these clones contain replication origins using an assay based on a 2D-gel analysis of in vivo-derived DNA (aim #3). In support of these aims, the principal investigator shows and impressive amount of preliminary progress. (1) DNA from human fibroblasts were labeled with BrdU (early replicating DNA should contain more BrdU). This material was fractionated (CsC1 density gradient) and cloned to give two libraries (ERC2 and ERC3 on grids), which contains less than 2 percent of the human genome. (2) Several approaches were taken to establish that the library does contain early replicating DNA. Several genes (c-myc, APRT...) are known to be early replicating, and the libraries were shown to contain these genes. DNA was labeled with BrdU, and the chromosomal bands where BrdU could be detected most often, were likely to contain early replicating DNA. Based on this analysis, six bands in the human genome (e.g., 15q22), which were most frequently observed, are the focus of this proposal. Several STSs (e.g., PML gene associated with a translocation in acute promyelocytic leukemia), which are known to be on band 15q22, were shown to be in the early replicating DNA libraries (p 32, Table III). (3) PML DNA was used to identify a clone from the ERC2 and ERC3 libraries. The clone maps appropriately (i.e., to the Q-arm of chromosome 15 by FISH). (4) 11kbp was sequenced from this clone, and, intriguingly, a region shows homologies to autonomously replicating sequences (ARSs), matrix attachment regions (MARS), a topo II binding site and pyrimidine runs (thought to be primase binding sites) (5) The principal investigator has also developed ways to enrich for DNA containing origins (column with higher affinity for ss-DNA, which origins should have), and has mastered a 2D electrophoresis method to detect unusual DNA structures that are formed during DNA replication (conceptually presented in Fig 18, p42).